动态压应力对大鼠干骺端软骨干细胞PTHrP mRNA表达的影响

Effects of dynamic pressure on the expression of PTHrP mRNA in metaphyseal cartilage stem cells of rats

目的研究动态压应力对大鼠干骺端软骨干细胞甲状旁腺激素相关蛋白(PTHrP)mRNA表达的影响,进一步探索纤维形肌动蛋白(F-actin)是否参与该力学信号转导过程。方法免疫磁珠法分离培养大鼠干骺端软骨干细胞。第3代大鼠干骺端软骨干细胞按照动态压应力强度的大小随机分为4组:0%、3%、6%、12%形变组,使用自行制备的动态压应力培养装置对各组细胞施加不同强度的压应力刺激24h,流式细胞术检测各组细胞周期分布及凋亡率,实时定量PCR检测各组细胞PTHrP mRNA表达水平。进一步,第3代大鼠干骺端软骨干细胞按照是否施加压应力及细胞骨架松弛素D随机分为4组:对照组、单纯压应力组(6%形变)、压应力+细胞骨架松弛素D组、单纯细胞骨架松弛素D组,各组细胞施加干预24h后以鬼笔环肽染F-actin纤维,置于激光共聚焦显微镜下观察,以及实时定量PCR检测各组细胞PTHrP mRNA表达水平。结果流式细胞术结果显示:0%、3%、6%、12%形变组细胞G0/G1、G2/M及S期比例差异无统计学意义(P>0.05);3%、6%形变组凋亡率与0%形变组相比,差异无统计学意义(P>0.05),12%形变组凋亡率明显高于对照组(P<0.05)。激光共聚焦显微镜观察结果表明:单纯压应力组F-actin纤维排列较对照组整齐,相互平行,与受力方向趋于一致;压应力+细胞骨架松弛素D组及单纯细胞骨架松弛素D组细胞内F-actin纤维结构破坏,相互聚集缠绕成团块状。实时定量PCR结果显示:3%形变组细胞PTHrP mRNA表达水平与0%形变组差异无统计学意义(P>0.05),6%及12%形变组细胞PTHrP mRNA表达量明显高于0%形变组(P<0.05);单纯压应力组细胞PTHrP mRNA表达量明显高于对照组(P<0.05),单纯细胞骨架松弛素D组细胞PTHrP mRNA表达量与对照组相比差异无统计学意义(P>0.05),压应力+细胞骨架松弛素D组细胞PTHrP mRNA表达量高于对照组,但低于单纯压应力组(P<0.05)。结论适当强度的动态压应力可以上调大鼠干骺端软骨干细胞PTHrP mRNA表达,在该力学信号转导过程中有F-actin的参与。

Objective To study the effect of dynamic pressure on the expression of parathyroid hormonerelated protein（PTHrP）mRNA in metaphyseal cartilage stem cells of rats so as to further explore whether fiber actin（F-actin）is involved in the mechanical signal transduction process.Methods We isolated and cultured metaphyseal cartilage stem cells of rats by immunomagnetic beads.The third-generation rat metaphyseal cartilage stem cells were randomly divided into four groups：0%,3%,6%,and 12% deformed groups according to the size of dynamic pressure strength.We used a self-prepared dynamic tonic culture device to exert different intensity of pressure on each group of cells for 24 hours.Flow cytometry was used to detect the cell cycle distribution andapoptosis rate.The expression of PTHrP mRNA in each group was detected by Real-time quantitative PCR.Furthermore,the third-generation rat metaphyseal cartilage stem cells were randomly divided into four groups：control group,simple pressure group（6% deformation）,pressure＋cytoskeleton relaxin D group,and simple cytoskeleton relaxin D group according to whether or not to apply pressure and cytoskeleton relaxin D.F-actin fibers in each group of cells were stained with phalloidin and placed under a laser scanning confocal microscope.The expression of PTHrP mRNA in each group was detected by Real-time quantitative PCR.Results The results of flow cytometry showed no significant difference in G0/G1,G2/M and S phases between 0%,3%,6% and 12%deformed groups（P〉0.05）.There was no significant difference in the apoptosis rate between 3% and 6%deformed groups compared with0% deformed group（P〉0.05）.The apoptosis rate was significantly higher in12%deformed group than in control group（P〈 0.05）.The results of laser confocal microscopy showed that the arrangement of F-actin fibers in the pressure group was neat and parallel compared with that in the control group,which was consistent with the direction of force.The intracellular F-actin fiber structure in pressure＋cytoskeleton relaxin D group and simple cytoskeleton relaxin D group was destroyed and aggregated into clusters.Real-time quantitative PCR results showed that PTHrP mRNA expression did not significantly differ between 3% and 0%deformed groups（P〉0.05）.The expression of PTHrP mRNA in 6% and 12% deformed groups was significantly higher than that in0% group（P〈0.05）.The expression of PTHrP mRNA in the cells of simple pressure group was significantly higher than that in the control group（P〈0.05）.There was no significant difference in the expression of PTHrP mRNA between simple cytoskeleton relaxin D group and control group（P〉0.05）.The mRNA expression of PTHrP was higher in pressure＋cytoskeleton relaxin D group than that in control group,but lower than in simple pressure group（P〈0.05）.Conclusion The dynamic pressure of proper intensity can increase the mRNA expression of PTHrP in chondrocytes of metaphyseal hypertrophy in rats,and F-actin is involved in the mechanical signal transduction process.